Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC50 values ranging from 6–9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.
References
[1]
Stanway G, Brown F, Christian P, Hovi T, Tyypia T, et al. (2005) Family Picornaviridae. In virus taxonomy. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA, editors. Eighth Report of the International Committee on Taxonomy of viruses. San Diego: Elsevier Academic Pres. pp. 757–778.
[2]
Brown BA, Pallansch MA (1995) Complete nucleotide sequence of Enterovirus 71 distinct from poliovirus. Virus Res 39: 195–205.
[3]
Shindarov LM, Chumakov MP, Voroshilova MK, Bojinov S, Vasilenko SM, et al. (1979) Epidemiological, clinical, and pathomorphological characteristics of epidemic poliomyelitis-like disease caused by Enterovirus 71. J Hyg Epidemiol Microbiol Immunol 23: 12.
[4]
Nagy G, Takatsy S, Kukan E, Mihaly I, Domok I (1982) Virological diagnosis of Enterovirus type 71 infections: experiences gained during an epidemic of acute CNS diseases in Hungary in 1978. Arch Virol 71: 217–227.
[5]
McMinn PC (2002) An overview of the evolution of Enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26: 91–107.
[6]
Chan YF, Sam IC, Wee KL, AbuBakar S (2011) Enterovirus 71 in Malaysia: A decade later. Neurol Asia 16: 1–15.
Ho M, Chen ER, Hsu KH, Twu SJ, Chen KT, et al. (1999) An epidemic of Enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med 341: 929–935.
[10]
Yang F, Ren L, Xiong Z, Li J, Xiao Y, et al. (2009) Enterovirus 71 outbreak in the People’s Republic of China in 2008. J Clin Microbiol 47: 2351–2352.
[11]
Yi L, Lu J, Kung HF, He ML (2011) The virology and developments toward control of human Enterovirus 71. Crit Rev Microbiol 37: 313–327.
[12]
Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, et al. (1998) Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 4: 1302–1307.
[13]
Matthews T, Salgo M, Greenberg M, Chung J, DeMasi R, et al. (2004) Enfuvirtide: the first therapy to inhibit the entry of HIV-1 into host CD4 lymphocytes. Nat Rev Drug Discov 3: 215–225.
[14]
Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, et al. (2008) A virocidal amphipathic a-helical peptide that inhibits Hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 105: 3088–3093.
[15]
Kim DH, Ni Y, Lee SH, Urban S, Han KH (2008) An anti-viral peptide derived from the preS1 surface protein of Hepatitis B virus. BMB Rep 41: 640–644.
[16]
Shih SR, Tsai MC, Tseng SN, Won KF, Shia KS, et al. (2004) Mutation in Enterovirus 71 capsid protein VP1 confers resistance to the inhibitory effects of pyridyl imidazolidinone. Antimicrob Agents Chemother 48: 3523–3529.
[17]
Sim AC, Luhur A, Tan TM, Chow VT, Poh CL (2005) RNA interference against Enterovirus 71 infection. Virology 341: 72–79.
[18]
Reed LJ, Muench H (1983) A simple method of estimating fifty per cent endpoints. The Am J Hyg 27: 493–497.
[19]
Tan EL, Yong LL, Quak SH, Yeo WC, Chow VT, et al. (2008) Rapid detection of Enterovirus 71 by real-time TaqMan RT-PCR. J Clin Virol 42: 203–206.
[20]
Jones JC, Turpin EA, Bultmann H, Brandt CR, Schultz-Cherry S (2006) Inhibition of Influenza virus infection by a novel antiviral peptide that targets viral attachment to cells. J Virol 80: 11960–11967.
[21]
Akkarawongsa R, Pocaro NE, Case G, Kolb AW, Brandt CR (2009) Multiple peptides homologous to Herpes Simplex Virus type 1 glycoprotein B inhibit viral infection. Antimicrob Agents Chemother 53: 987–996.
[22]
Tiwari V, Liu J, Valyi-Nagy T, Shukla D (2011) Anti-heparan sulfate peptides that block Herpes Simplex Virus infection in vivo. J Biol Chem 286: 25406–25415.
[23]
Liu R, Tewari M, Kong R, Zhang R, Ingravallo P, et al. (2010) A peptide derived from Hepatitis C virus E2 envelope protein inhibits a post-binding step in HCV entry. Antiviral Res 86: 172–179.
[24]
Hrobowski YM, Garry RF, Michael SF (2005) Peptide inhibitors of Dengue Virus and West Nile Virus infectivity. Virol J 2: 49.
[25]
Li C, Wang H, Shih SR, Chen TC, Li ML (2007) The efficacy of viral capsid inhibitors in human enterovirus infection and associated diseases. Curr Med Chem 14: 847–856.
[26]
Chen P, Song Z, Qi Y, Feng X, Xu N, et al. (2012) Molecular determinants of Enterovirus 71 viral entry: a cleft around Q172 on VP1 interacts with a variable region on scavenge receptor B 2. J Biol Chem 287: 6406–6420.
[27]
Liu J, Thorp SC (2002) Cell surface heparan sulfate and its roles in assisting viral infections. Med Res Rev 22: 1–25.
Mann DM, Romm E, Migliorini M (1994) Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin. J Biol Chem 269: 23661–23667.
[30]
Shimazaki K, Tazume T, Uji K, Tanaka M, Kumura H, et al. (1998) Properties of a heparin-binding peptide derived from bovine lactoferrin. J Dairy Sci 81: 2841–2849.
[31]
Van der Strate BW, Beljaars L, Molema G, Harmsen MC, Meijer DK (2001) Antiviral activities of lactoferrin. Antiviral Res 52: 225–239.
[32]
Marchetti M, Trybala E, Superti F, Johansson M, Bergstrom T (2004) Inhibition of Herpes Simplex Virus infection by lactoferrin is dependent on interference with the virus binding to glycosaminoglycans. Virology 318: 405–413.
[33]
Weng TY, Chen LC, Shyu HW, Chen SH, Wang JR, et al. (2005) Lactoferrin inhibits Enterovirus 71 infection by binding to VP1 protein and host cells. Antiviral Res 67: 31–37.
[34]
Heinz BA, Vance LM (1995) The antiviral compound enviroxime targets the 3A coding region of Rhinovirus and Poliovirus. J Virol 69: 4189–4197.
[35]
Pevear DC, Tull TM, Seipel ME, Groarke JM (1999) Activity of pleconaril against Enteroviruses. Antimicrob Agents Chemother 43: 2109–2115.
[36]
Li ZH, Li CM, Ling P, Shen FH, Chen SH, et al. (2008) Ribavirin reduces mortality in Enterovirus 71-infected mice by decreasing viral replication. J Infect Dis 197: 854–857.
[37]
Patick AK, Ford C, Binford S, Fuhrman S, Brothers M, et al. (1997) Evaluation of the antiviral activity and cytotoxicity of peptide inhibitors of Human Rhinoviurs 3C protease, a novel target for antiviral intervention. Abstracts of the 10th International Conference on Antiviral Research: Antiviral Res. A75 p.
[38]
Rotbart HA, O’Connell JF, McKinlay MA (1998) Treatment of Human Enterovirus infections. Antiviral Res 38: 1–14.
[39]
Lin TY, Chu C, Chiu CH (2002) Lactoferrin inhibits Enterovirus 71 infection of human embryonal rhabdomyosarcoma cells in vitro. J Infect Dis 186: 1161–1164.
[40]
Wu KX, Ng MM, Chu JJ (2010) Developments towards antiviral therapies against Enterovirus 71. Drug Discov Today 15: 1041–1051.
[41]
Huther A, Dietrich U (2007) The emergence of peptides as therapeutic drugs for the inhibition of HIV-1. AIDS Rev 9: 208–217.
[42]
Gropp R, Frye M, Wagner TO, Bargon J (1999) Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy. Hum Gene Ther 10: 957–964.
[43]
Andersen JH, Osbakk SA, Vorland LH, Traavik T, Gutteberg TJ (2001) Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts. Antiviral Res 51: 141–149.
[44]
Bastian A, Schafer H (2001) Human alpha-defensin 1 (HNP-1) inhibits adenoviral infection in vitro. Regul Pept 101: 157–161.
[45]
Belaid A, Aouni M, Khelifa R, Trabelsi A, Jemmali M, et al. (2002) In vitro antiviral activity of dermaseptins against herpes simplex virus type 1. J Med Virol 66: 229–234.
[46]
Andersen JH, Jenssen H, Gutteberg TJ (2003) Lactoferrin and lactoferricin inhibit Herpes simplex 1 and 2 infection and exhibit synergy when combined with acyclovir. Antiviral Res 58: 209–215.
